Introduction

Vertebrate telomeres are composed of TTAGGG tandem repeats that recruit the shelterin complex, which protects chromosome ends, regulates telomere length, recombination, and DNA damage checkpoints (1, 2). Shelterin is composed of TRF1, TRF2, POT1, TPP1, TIN2, and RAP1 (2). TRF1 and its paralog TRF2 bind to double-stranded TTAGGG repeats and interact with POT1 via interaction with TIN2 and TPP1 (2–4). POT1 interacts with single-stranded TTAGGG repeats in the telomeric 3′ overhang and, together with TRF1 and TRF2, ensures the protection of chromosome ends by repressing DNA damage signaling by the ATR and ATM kinases (5–7). RAP1 binds to telomeres via its interaction with TRF2 (8). TRF1 has been shown to contribute to telomere length regulation and suppresses DNA breakage at TTAGGG repeats under replicative stress, a phenomenon described as telomere fragility (5, 6, 9, 10). Consistent with this, loss of TRF1 results in telomere replication errors and the activation of ATR signaling in S-phase (5, 6). Recently, evidence is accumulating that links altered shelterin function with human cancer. POT1 mutations have been linked to telomeric and chromosomal abnormalities in chronic lymphocytic leukemia (11). Wnt/β-catenin signaling was shown to drive TRF2 expression, improving telomere function in cancer cells (12). In addition, TRF2 protein levels were shown to be controlled by the p53-inducible E3 ubiquitin ligase Siah1, linking shelterin function with the p53 tumor suppressor pathway (13). Importantly, mice deficient for TRF1 display telomere fragility and increased cancer formation in the absence of p53. This indicates that reduced TRF1 expression can enhance cancer formation by driving telomere fragility and genomic instability (6). Together, this suggests that shelterin function is intertwined with central pathways in tumorigenesis and tumor suppression. miRNAs are 20–23 nucleotide, single-stranded RNA molecules that control gene expression by reducing the translation or stability of target mRNAs (14). Extensive studies demonstrated that miRNAs control the expression of crucial tumor suppressors or oncogenes and propagate essential features of cancer progression (15). Importantly, miRNA expression signatures have been established as efficient prognostic and predictive biomarkers, underlining the clinical relevance of miRNAs (15). Although telomere function is a central aspect in cancer and aging, miRNAs that modulate the expression of shelterin components are to date not known. Of interest, a recent study links miR-138 with telomerase (TERT) expression; however, the impact of miR-138 on telomere homeostasis is not known (16).

Here, we performed a small-scale screen to identify clinically relevant miRNAs that modulate telomere function in human breast cancer by targeting the expression of TRF1. We found that the oncomiR miR-155 targets a conserved sequence motif in the 3′UTR of TRF1, resulting in reduced protein expression. miR-155 is efficiently upregulated in human breast cancer specimen, correlates with reduced TRF1 protein levels, and is linked with poor prognosis in estrogen receptor (ER)–positive breast cancer. On the mechanistic level, we demonstrate that miR-155-dependent reduction of TRF1 expression results in telomere elongation, increased telomere damage, increased telomere fragility, and chromosome instability. In contrast, reducing miR-155 expression improves telomere function and genomic stability. Together, our data identify miR-155 as clinically relevant, novel telomere regulator that drives telomere fragility and genomic instability by repressing TRF1 expression. Our work introduces miRNAs as modulators of shelterin function, anticipating the existence of additional “telo-miRNAs” that link telomere function with fundamental processes in telomere-related diseases such as cancer and organismal aging.

Telomere DNA FISH

Preparation of metaphases and telomere DNA FISH was performed as previously described (17). At least 20 metaphases were analyzed for metaphase chromosome aberrations (TFL-TELO software). For quantitative telomere DNA FISH analysis, at least 20 interphase nuclei were analyzed using spot IOD analysis (TFL-TELO) software. The Student t test was used to calculate statistical significance.

Immunofluorescence combined with telomere DNA FISH

For immunofluorescence, cells were fixed in 4% PFA, followed by treatment with 0.1% Triton X-100 in 1× PBS for 10 minutes at room temperature. Cells were blocked for 30 minutes in 3%BSA (1× PBS) and incubated with a mouse anti-phospho-γH2A.X (S139) antibody (clone JBW301, Millipore, 06-570) in 3% BSA, 0.1% Tween-20 at room temperature for 1 hour. After incubation with secondary antibodies, cells were fixed in 1% PFA and subjected to standard telomere DNA FISH (18). Nuclei showing at least three telomere-γH2AX colocalization events were considered for the quantification (minimum 40 nuclei). The Student t test was used to calculate statistical significance.

miR-155 alters telomere structure and function

We next wished to link miR-155 to molecular pathways that modulate TRF1-related aspects of telomere function. To test whether alteration of miR-155 expression levels does not only impact on global TRF1 expression levels but also alter TRF1 abundance at telomeres, we transiently transfected SK-BR-3 luminal breast cancer cells with mimic-miR-155, antago-miR-155 siRNAs, or TRF1-specific siRNAs and performed quantitative immunofluorescence analysis using anti-TRF1 antibodies. We found that ectopically introduced miR-155 significantly reduced fluorescence signal intensity, indicative for a reduced abundance of TRF1 at telomeres (Fig. 4A). This result is also reflected by the appearance of an increased proportion of telomeres with low TRF1 loading; this effect was recapitulated by transfecting a TRF1-specific siRNA pool (Fig. 4A). In contrast, treatment with antago-miR-155 increased TRF1 abundance at telomeres (Fig. 4A). Same results were obtained when modulating miR-155 levels in H1299 non–small lung carcinoma cells, that express elevated levels of TRF1 and miR-155 compared with breast cancer cell lines used in this study (Fig. 1A and Supplementary Fig. S4A and S4B). In line with Western blotting results we found that alteration of miR-155 levels does not impact on the abundance of TRF2 or POT1 at telomeres (Fig. 2D and E and Supplementary Fig. S4C and S4D). Our results indicate that miR-155-dependent regulation of TRF1 expression is an efficient mechanism to control the abundance of TRF1 at telomeres. TRF1 acts as negative regulator of telomere length, presumably by restricting the access of telomerase to chromosome ends (9, 10). In line with this, we found a significant increase in telomere length after 3 cycles of transient transfection of telomerase-positive SK-BR-3 cells with mimic-miR-155 siRNAs or TRF1-specific siRNAs (Fig. 4B). This result was recapitulated when TRF1 expression was reduced by the stable overexpression of miR-155 in long-term experiments using SK-BR-3 cells (Supplementary Fig. S5A–S5E). This indicates that miR-155 promotes telomere elongation by reducing abundance of TRF1 at telomeres. Loss of TRF1 is associated with impaired telomere function, leading to the elicitation of a DNA damage response at telomeres (5, 6). In line with this, we found by Western blotting that transient transfection of luminal MCF-7 and SK-BR-3 breast cancer cell lines with mimic-miR-155 siRNAs result in elevated levels of γH2AX, indicative for the activation of a DNA damage response (Fig. 4C and D, left). DNA damage response was associated with the activation of wild-type p53 in MCF-7 cells, as indicated by increased levels of phospho-p53 (Ser15) (Fig. 4C and D, left). In SK-BR-3 cells, which express mutant p53, introduction of miR-155 was not able to significantly increase phosphorylation of p53. This is presumably due to the high basal levels of phospho-p53 (Ser15) present in this cell line (Fig. 4D). In line with results from TRF1 knockout mice, we found that miR-155-dependent reduction of TRF1 expression leads to the phosphorylation of ATM and ATR and the downstream DNA damage checkpoint kinases Chk1 and Chk2 (Fig. 4C and D, right). To test whether telomere dysfunction contributes to the increased DNA damage load in the context of ectopic miR-155, we performed γH2AX immunofluorescence combined with telomere DNA FISH. Consistent with a proposed role in controlling telomere protection, we found that ectopic introduction of miR-155 increased the abundance of γH2AX localized at telomeres in MCF-7 and SK-BR-3 cells (Fig. 4E and F). This effect was recapitulated by RNAi-mediated depletion of TRF1 and in long-term experiments using MCF-7 cells stably overexpressing miR-155 (Supplementary Fig. S6A–S6D). Importantly, introduction of antago-miR-155 reduces basal levels of telomere dysfunction in MCF-7 and SK-BR-3, indicating that chromosome end protection is miR-155 dosage sensitive (Fig. 4E and F). Together, these data indicate that miR-155 affects telomere length regulation and telomere capping by modulating the expression of TRF1.

miR-155 drives alterations in telomere function in breast cancer cell lines. A, exemplary images of fragile telomeres, characterized by multiple telomere signals; for enlargement see images 1 and 2. B, frequency of telomere fragility in SK-BR-3 cells transfected with mimic-miR-155 or antago-miR-155 siRNAs. C, telomere fragility frequency after combined transfection of SK-BR-3 cells with mimic-miR-155 and TRF1 siRNAs (top). Bottom, TRF1 levels in experimental samples. Actin was used as a loading control. D, anti-TRF1 Western blotting of SK-BR-3 cells cotransfected with Flag-tagged TRF1 and mimic-miRNAs molecules; actin was used as loading control. E, telomere fragility in cells described in D; fragility induced by miR-155 is rescued by cointroduction of Flag-tagged TRF1. F, telomere fragility in antago-miR-155-transfected SK-BR-3 cells treated or untreated with Aphidicolin. G, increased frequency of telomeric sister chromatid fusions induced by miR-155 is rescued by cointroduction of Flag-tagged TRF1 in SK-BR-3 cells (cells are described in D). Exemplary images are shown. H, telomere sister chromatid fusions in antago-miR-155–transfected SK-BR-3 cells treated or untreated with Aphidicolin. N, number of metaphases spreads analyzed; n, number of analyzed chromosomes. At least three experimental replicas were analyzed. A Student t test was used to calculate statistical significance. P values are shown.

Altogether, we show that miR-155-dependent regulation of TRF1 is an efficient mechanism to control telomere fragility and genomic stability in human breast cancer cells. The consistent upregulation of miR-155 in breast cancer suggests that impaired telomere function is a central aspect of the oncogenic function of miR-155 that promotes genomic instability in human breast cancer and contributes to reduced distant metastasis-free survival and relapse-free survival in ER+ breast cancer.

Discussion

Telomere dysfunction is a major type of chromosome damage in cancer and interfering with regulators of telomere function drives central features of cancer such as loss of heterozygosity, chromosomal rearrangements, aneuploidy, and the repression of DNA damage response checkpoints (33, 34). The importance of telomere regulation in human cancer is underlined by the upregulation of telomerase activity in 90% of human cancers and frequent alteration of the expression levels of shelterin components (35–43). Increased cancer formation upon loss of TRF1 in the context of compromised tumor suppression suggests that alteration of TRF1 expression drives genomic instability, a hallmark feature of human cancer (6, 11). Using a small-scale miRNA screening approach, we show that miR-155 efficiently regulates TRF1 expression by targeting a partially conserved sequence motif in the TRF1 3′UTR. miR-155 is a classic onco-miRNA that is processed from a noncoding transcript encoded by the BIC locus and has been linked to lymphomagenesis (44, 45). However, more recent studies indicate a general role for miR-155 in human cancer, as demonstrated by a robust upregulation in breast, colon, and lung cancer (46). Ectopic miR-155 was shown to drive the proliferation of breast cancer cell lines in vitro but also, when xenografted into nude mice, to underline the role of miR-155 as onco-miRNA in breast cancer (47, 48). In addition to its role in promoting cell proliferation, a large panel of targets has been identified that link miR-155 to cancer relevant pathways, including apoptosis, inflammation, or DNA mismatch repair (22, 23, 49). miRNA expression signatures have been established as efficient prognostic and predictive biomarkers, underlining the clinical relevance of miRNAs (15).

Here, we found that elevated miR-155 levels reduce TRF1 abundance at telomeres and competing endogenous miR-155 expression increases TRF1 at telomeres. This indicates that alterations in miR-155 expression do not only affect the nuclear pool of TRF1, but directly impact on TRF1 abundance at telomeres. miR-155-dependent modulation of the stoichiometry of shelterin complex components resulted in impaired telomere function related to reduced TRF1 expression. We show that miR-155 is a positive regulator of telomere length in telomerase-positive SK-BR-3 a luminal breast adenocarcinoma-derived cell line. This is in line with previous results from TRF1 gain- and loss-of-function experiments (10, 50). Of note, miR138 has been proposed to regulate the expression of human telomerase; however, the relevance of this interaction for telomere homeostasis remains unaddressed (16). In line with reports demonstrating a central role of TRF1 in controlling the replication of telomeres, we show that elevated miR-155 levels promote telomere fragility coupled with the recruitment of the DNA damage marker γH2AX at telomeres (5, 6, 32). Importantly, miR-155-dependent telomere fragility resulted in increased genomic instability exemplified by an increased telomere sister chromatid fusions in SK-BR-3 cells that are characterized by impaired tumor suppression due to the expression of a mutant form of p53. In line with this, experimental reduction of endogenous miR-155 levels improves genomic stability at telomeres. These findings are in line with increased cancer occurrence in TRF1 knockout mice lacking p53 (6). Our results show that driving telomere fragility and genomic instability is a central aspect in the repertoire of cancer-promoting functions of miR-155. This result is of special relevance in the light of the recent finding that more than 50% of recurrent amplifications/deletions in human diffuse large B-cell lymphoma map to fragile sites (29). We show that increased miR-155 expression correlates with reduced TRF1 protein levels in specimen from luminal breast cancer that is typically ER+. Importantly, low TRF1 expression is an independent predictor of clinical outcome that is associated with reduced distant metastasis-free survival and relapse-free survival in patients with ER+ breast cancer. This pattern is recapitulated by a panel of validated miR-155 targets, underlining the role of miR-155 as “oncomiR.” However, this also indicates that TRF1 expression is an integral component of a miR-155-dependent signature that predicts poor clinical outcome in ER+ breast cancer.

In conclusion, our work identifies the “oncomiR” miR-155 as the first miRNA that controls the expression of a shelterin component to alter telomere function. Our finding that promotion of telomere-related genomic instability by miR-155 is linked with poor clinical outcome in an ER+ breast cancer underlines the relevance of mechanisms that control telomere function during cancer formation and/or progression.

Our work also anticipates the existence of multiple miRNAs that affect telomere function and homeostasis. Identification and functional characterization of “telo-miRNAs” is expected to provide inroads into the understanding and potential therapeutic treatment of telomere-related maladies such as cancer and aging.

Grant Support

This work was supported by the Italian Association for Cancer Research (AIRC) grant cod. 10299, a Fondazione Veronesi grant (S. Schoeftner), and a Young Investigator Grant, Ministry of Health GR-2007-683407 (R. Benetti).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).